这项研究得到了美国国立卫生研究院的支持, 该计划的机构间协议 AOD13015-001。我们要感谢斯蒂芬妮 Froberg 和彼得赫斯特在视频制作方面的努力和专长。

1. 细胞培养维护
<ol><li>生长细胞线 SV40-HCEC 在37°c, 5% CO2, 90% 湿度 DMEM F-12 与15% 胎牛血清 (血清), 1% l-谷氨酰胺, 10 µg/升表皮生长因子 (EGF), 5 毫克/升胰岛素。</li>
	<li>通过细胞线每3到4天 (取决于播种密度), 以确保融合从来没有超过80% 在文化维护。</li>
	<li>使用分离溶液 (每150厘米2瓶) 将细胞从烧瓶中取出, 并在37摄氏度处孵化不超过8分钟 (14 毫升溶液)。</li>
	<li>用相同容积的细胞培养培养基中和分离液, 在 160 x g 处离心7分钟, 将50毫升锥形管中的细胞颗粒化。</li>
	<li>在培养基中重新悬浮细胞颗粒 (每150厘米2瓶20毫升)。</li>
	<li>使用自动手持单元格计数器和新烧瓶中的种子进行计数, 使其在不超过80% 融合的情况下生长3到4天。</li>
</ol>2. 实验用平板细胞
<ol><li>用相衬光镜检查 SV40-HCECs 的烧瓶, 以确保融合小于 80%, 并评估一般细胞的健康状况。</li>
	<li>分离, 颗粒, 并用重悬和计数 SV40-HCECs 从烧瓶中所述的步骤1.3 至1.6 的细胞培养维护。使用含有0.5 µg/毫升松 (HCORT 培养基) 的 SV40-HCEC 培养基并用重悬细胞。</li>
	<li>在一次性培养基瓶中, 在预热后的 HCORT 培养基中, 准备悬浮 SV40-HCECs 17857 细胞/毫升。<ol>
<li>为要播种的盘子数量准备足够数量的细胞悬浮。</li>
		<li>种子至少 14 x 96-井板与细胞为每个 siRNA 库板块进行研究。</li>
	</ol>
</li>
	<li>旋转瓶子均匀悬浮细胞, 将足够数量的细胞悬浮液倒入一个自动液体处理器的轨道振动筛巢中的储层, 并将含有细胞悬浮物的瓶子贮存在37摄氏度暖板上。过程。</li>
	<li>使用自动液体处理程序将单元格添加到每井1000年细胞密度 (5000 个细胞/厘米2) 的板上, 在70µL 的96井板中。对所有步骤使用50µL/秒吹打速度。<ol>
<li>在播种过程中不断地运行 100 rpm 的轨道振动筛。</li>
		<li>使用自动液体处理器将电池悬浮液混合三次 (140 µL 混合容积)。</li>
		<li>吸入140µL 细胞悬浮液, 并将70µL 放入两个细胞培养板的每一个井中。</li>
		<li>重复步骤2.5.3 和 2.5.4, 直到所有的盘子都被细胞所播种。</li>
		<li>在播种过程中需要重新填充细胞悬浮液。</li>
	</ol>
</li>
	<li>在将盘子播种后, 将它们从自动液体处理器中取出, 在室温下孵化30分钟, 然后将它们转移到细胞培养孵化器。</li>
	<li>第二天早上, 用一个自动成像系统在细胞培养孵化器中评估每块井的细胞密度, 并排除研究中没有15% 和22% 汇合的内部井的任何板块。</li>
</ol>3. 染细胞与 siRNA
<ol><li>从供应商处获取 siRNA 库板, 预配置为包含每板 80 siRNA 目标 (见图 4), 列1和12为空。</li>
	<li>根据制造商的说明19, 将 siRNA 库板与 siRNA 缓冲器重建为 2 pmol/µL 的最终浓度。</li>
	<li>染24小时后, 4 pmol/井 siRNA 和0.3 µL/井转染试剂。按照转染试剂制造商的协议执行所有步骤 (参见图 4中的板布局)20。<ol>
<li>使用自动液体处理程序执行所有 transfections, 并对所有步骤使用25µL/秒吹打速度。使用预配置的提示框只处理将被转染的水井。</li>
		<li>使用库板的上半部分染一组六个被称为 "顶部板集" 的复制板 (每 siRNA 六个复制, n=6)。</li>
		<li>使用库板的下半部分染不同的六个复制板, 称为 "底板集" (每 siRNA 六个复制, n=6)。</li>
		<li>使用 "全盘集" 一词来描述已转染图书馆 siRNA 的12个细胞板块。</li>
		<li>染井 B2-B6 所有板块的全板组与负池 siRNA 后, 所有顶部和底部板集已转染图书馆 siRNA。</li>
		<li>也染井 B2-B6 另外两个板材, 不接收图书馆 siRNA 并且将担当未曝光的控制 (一为顶部板集和一个为底部板设置), 与消极水池 siRNA。</li>
	</ol>
</li>
	<li>在细胞培养孵化器中孵化所有细胞板块4小时后, 所有的转染混合添加和混合通过温和的攻丝每小时。</li>
	<li>使用自动液体处理程序冲洗两次板的所有水井, 预热 HCORT 介质稀释1:5 在 PBS。对所有步骤使用50µL/秒吹打速度。<ol>
<li>从每个井中抽出100µL, 并将其丢弃在废物库中。</li>
		<li>添加到每个井100µL/井预热 HCORT 介质稀释1:5 在 PBS, 这是包含在一个不同的水库在足够数量的板块数量。</li>
		<li>对每个盘子重复步骤3.5.1 和3.5.2。<ol>
<li>根据需要清空废物库。</li>
			<li>根据需要, 在 PBS 中 HCORT 介质稀释1:5。</li>
		</ol>
</li>
		<li>从每个井中抽出100µL, 并将其丢弃在废物库中。</li>
	</ol>
</li>
	<li>Refeed 所有井的板材与100µL 或好预热的 HCORT 媒介, 在一个不同的水库包含在一个充足的容量介质为板材的数量。<ol>
<li>在需要的介质中重新填充储层。</li>
	</ol>
</li>
	<li>将所有板块的井 C2-C6 为未转染的控制。</li>
	<li>保持水井 B7-B11 和 C7-C11 的所有板块未转染, 用作药物和车辆控制毒物暴露。</li>
</ol>4. 第二天 Refeed 细胞
<ol><li>使用自动液体处理程序 refeed 所有油井的板材。对所有步骤使用50µL/秒吹打速度。<ol>
<li>从每个井中抽出100µL, 并将其丢弃在废物库中。</li>
		<li>Refeed 每个井与100µL/井预热 HCORT 介质, 这是包含在不同的水库, 并有足够的介质数量的板块是 refed。</li>
		<li>根据需要重复步骤4.1.1 和 4.1.2 refeed 所有细胞板。<ol>
<li>根据需要清空废物库。</li>
			<li>在需要的介质中重新填充储层。</li>
		</ol>
</li>
	</ol>
</li>
</ol>5. 积极控制加法
<ol><li>转染两天后, 暴露前两个小时, 准备一个62.5 µM 解决方案的积极控制小豆蔻明在 HCORT 介质中用于高放暴露和增加 B 排的8行稀释水库。<ol>
<li>对于 CP 曝光, 准备和使用 SKF 86002 的25µM 溶液。</li>
		<li>准备一个足够数量的正控制量, 以测试的板材数量。</li>
	</ol>
</li>
	<li>在 HCORT 介质 (车辆控制) 上准备0.625% 的亚甲基亚砜溶液, 并加入同一储层的 C 排。<ol>
<li>对于 CP 暴露, 准备和使用0.5% 溶液的亚砜。</li>
		<li>准备足够的车辆控制量的车牌数量的测试。</li>
	</ol>
</li>
	<li>使用自动液体处理程序添加正面和车辆控制, 以井 B7-B11 和 C7-C11 的每个细胞板, 并使用50µL/秒吹打速度的所有步骤。使用预先配置的提示框, 只处理将接收到正面和车辆控制的水井。<ol>
<li>从 B7-B11 和 C7-C11 中去除每个细胞板的10µL 介质, 将其丢弃到8排稀释油藏的 G 和 H 行中。</li>
		<li>将正面和车辆控制的10µL 转移到井中, 混合3次。</li>
	</ol>
</li>
	<li>把这些电池板还给孵化器。</li>
	<li>重复步骤5.3 至 5.4, 直到所有电池板都收到正面和车辆控制。</li>
</ol>6. 培养细胞的 HF 暴露
注意: 高放具有腐蚀性和剧毒。
<ol><li>使用双腈手套, 实验室涂层, 一次性聚乙烯套筒保护器和安全眼镜, 在化学油烟机中执行所有化学接触操作。<ol>
<li>获取高放作为48% 解决方案。</li>
		<li>稀释高放到1% 与超纯水和存储在5毫升整除数10毫升厚壁聚乙烯瓶, 以提高安全性。</li>
		<li>在危险废物流处置之前, 对与高放和任何残留液体高放有2.5% 葡萄糖酸钙接触过的吸管提示和水库进行净化。</li>
	</ol>
</li>
	<li>对于每个 siRNA 库板块, 请在150毫升瓶中加入288µL 1% 高放到80毫升预热 HCORT 培养基, 准备一个0.0036% 高放介质溶液。<ol>
<li>旋流瓶和孵化稀释高放在37°c 孵化器在化学油烟机10分钟。</li>
	</ol>
</li>
	<li>通过旋转瓶子, 再将高放介质溶液混合, 并将高放介质溶液添加到板上较暖和的试剂库中。</li>
	<li>与两名技术员一起工作, 进行接触。<ol>
<li>让技术员在正确的100µL 从每一个细胞板的内部井使用12通道 pipettor, 然后把盘子传给技术员在左边。</li>
		<li>在左边的技术员增加100µL 每井高放中等解答。</li>
		<li>重复步骤6.4.1 和6.4.2 所有的细胞板, 将暴露于毒物。</li>
		<li>还重复步骤6.4.1 和6.4.2 未曝光的控制板, 利用新鲜的 HCORT 介质, 而不是高放介质溶液。</li>
	</ol>
</li>
	<li>将板块放在化学油烟机孵化器中20分钟, 然后将它们归还细胞培养孵化器。</li>
	<li>重复正面控制加法步骤 (步骤5.1 至 5.5), 如前所示, 一旦所有板块被暴露, 并返回到细胞培养孵化器。</li>
</ol>7. 培养细胞的 CP 暴露
注意: CP 是剧毒和刺激性。
<ol><li>使用双腈手套, 实验室涂层, 一次性聚乙烯套筒保护器和安全眼镜, 在化学油烟机中执行所有化学接触操作。<ol>
<li>获取 CP。</li>
		<li>稀释 CP 到5% 在亚砜和存储在10毫升整除数10毫升闪烁瓶, 以提高安全性。</li>
		<li>在危险废物流中处置之前, 用2.5% 亚硫酸氢钠与 cp 和任何残留液体 cp 接触的吸管尖和储层进行净化。</li>
	</ol>
</li>
	<li>准备足够数量的预热 HCORT 培养基, 含1x 支笔/链球菌和预热的 PBS, 以供暴露的板材数量。</li>
	<li>对于每个顶板设置或底部板集, 添加8.04 µL 5% CP 在亚砜到50毫升整除预热 HCORT 培养基和混合良好的最终 CP 浓度0.0008%。将管子紧紧地盖住, 在化学油烟机的37°c 孵化器中孵化出1小时的溶液。<ol>
<li>时间增加 cp 到50毫升整除数的介质, 使每个暴露的顶板设置和底部板集收到毒物恰好1小时后, CP 被添加到50毫升的整除培养基。</li>
	</ol>
</li>
	<li>从细胞培养孵化器中取出一个顶板和1个未曝光的控制板, 并将它们放在靠近孵化期结束的化学油烟罩中。</li>
	<li>混合 CP 溶液通过反转管, 然后醒酒它到试剂库在1小时的孵化期结束。</li>
	<li>与两名技术员一起工作, 进行接触。<ol>
<li>让技术员在正确的100µL 从每一个细胞板的内部井使用12通道 pipettor, 然后把盘子传给技术员在左边。</li>
		<li>让技术人员在左侧添加100µL 的 CP 介质解决方案。</li>
		<li>重复步骤7.6.1 和7.6.2 的所有细胞板块的顶部板设置要暴露于毒物。</li>
		<li>还重复步骤7.6.1 和7.6.2 未曝光的控制板, 利用新鲜的 HCORT 介质, 而不是 CP 介质溶液。</li>
	</ol>
</li>
	<li>将盘子放在化学油烟机的37摄氏度孵化器中, 10 分钟。</li>
	<li>添加足够数量的 PBS 和 HCORT 培养基, 含有1x 笔/链球菌, 以试剂库近10分钟孵化期结束。</li>
	<li>使用12通道 pipettor 从单元格中取出 CP 解决方案, 并在10分钟孵化期结束时将其替换为 PBS 的100µL。</li>
	<li>立即从细胞板中取出 PBS, 用含有1x 笔/链球菌的 HCORT 介质中的100µL 来替换它。</li>
	<li>对于未曝光的控制板, 也重复步骤7.9 和7.10。</li>
	<li>将电池板返回标准细胞培养孵化器。</li>
	<li>对底部板集重复步骤7.3 到7.12。</li>
	<li>重复的积极控制添加步骤 (步骤5.1 至 5.5), 如前所述, 一旦所有板块已经暴露, 并返回到细胞培养孵化器。</li>
</ol>8. 样品收集和细胞活性测定
<ol><li>暴露后二十四小时, 在 PBS 中准备0.5 毫克/毫升 MTT 基板的溶液, 含10克/升葡萄糖, 暖至37摄氏度21。为每个板材准备10毫升的基板进行化验。</li>
	<li>对于要测定的板材数量, 在自动液体处理程序中添加足够数量的 MTT 基板溶液到储层上。</li>
	<li>使用自动液体处理程序收集样本, 并添加 MTT 基板使用50µL/秒吹打速度为所有步骤。进行预配置提示框, 以解决只有那些水井要化验。<ol>
<li>从细胞板内部60井中抽出95µL, 并将42.5 µL 存入两个384阱的储存板中。</li>
		<li>立即增加100µL 每井的 MTT 基质溶液的细胞板块, 并孵化他们在37°c 在细胞培养孵化器1.5 小时。</li>
		<li>根据需要, 用 MTT 衬底溶液重新填充储层。</li>
	</ol>
</li>
	<li>在细胞培养孵化器中孵育37摄氏度的细胞板块, 1 小时。</li>
	<li>密封384井存储板, 并存储在-80 °c, 以供后续分析。</li>
	<li>对所有顶部和底部板集以及相关的未公开控件重复步骤8.3 到8.5。<ol>
<li>在复制之间重复使用提示 (如果需要), 但在添加 MTT 基板解决方案和在板集之间切换时清洗它们。</li>
	</ol>
</li>
	<li>在1小时孵化后, 在自动液体处理程序中添加一个足够数量的亚砜到一个水库。</li>
	<li>使用自动液体处理程序添加亚砜, 并对所有步骤使用50µL/秒吹打速度。<ol>
<li>从每个单元的内部井中抽出100µL, 并将 MTT 基板溶液丢弃在废弃的储层中。<ol>
<li>根据需要清空废物库。</li>
		</ol>
</li>
		<li>覆盖每个井与100µL 亚砜。<ol>
<li>根据需要重新填充亚砜水库。</li>
		</ol>
</li>
	</ol>
</li>
	<li>在平板振动筛上摇动盘子3分钟。</li>
	<li>用平板光谱仪测量570和 690 nm 的吸光度。</li>
	<li>通过将暴露的吸光度值除以未曝光的控件, 减去背景并计算% 细胞的生存能力。使用平均未公开的负池 siRNA 控制来计算所有目标的% 细胞生存能力。</li>
</ol>9. 测定细胞培养中的 IL-8 浓度上清液
<ol><li>根据制造商的说明22, 测量细胞培养上清液中 IL-8 的浓度。创建一个检测板布局, 以适应样品和标准曲线。</li>
	<li>从-80 °c 冷藏柜中取出384井的储物板, 室温解冻, 并简单地将板材离心在油井底部收集样品。</li>
	<li>对于要运行的检测板的数量, 在试剂盒中制作足够数量的 anti-IL-8 受体珠和生物素化 anti-IL-8 抗体。使用50µL 的 anti-IL8 受体珠和50µL 生物素化 anti-IL8 抗体的比例9.9 毫升的检测缓冲器。<ol>
<li>使用多通道 pipettor, 添加珠/抗体混合物的黑色384井存储板。<ol>
<li>根据检测板布局, 将受体珠/抗体添加到用于样品和标准曲线的井中。</li>
			<li>为要运行的检测板数量添加足够数量的每井容积。</li>
		</ol>
</li>
	</ol>
</li>
	<li>用含有354µM 氯化钠的细胞培养培养基重建 IL-8 标准到 1000 pg/毫升。为要运行的检测板数量制作足够的容积。</li>
	<li>制作标准曲线的八个双重串行稀释。</li>
	<li>根据检测板的布局, 将标准曲线加成三个, 在黑色384井存储板的适当井中加入。为要运行的检测板数量添加足够数量的每井容积。<ol>
<li>同样, 添加含有354µM 氯化钠的细胞培养培养基, 不 IL-8 背景测量。</li>
	</ol>
</li>
	<li>使用自动液体处理程序来执行化验。使用预配置的提示框只处理由检测板布局指定的水井。对所有步骤使用50µL/秒吹打速度。<ol>
<li>将受体珠抗体混合物的8µL 转移到白色384井浅层测井板中。<ol>
<li>根据检测板的布局, 仅添加到指定样品和标准曲线的井中。</li>
		</ol>
</li>
		<li>根据检测板布局, 将标准曲线的2µL 转移到浅层试片的适当井中。</li>
		<li>准备一个3.54 米氯化钠溶液, 并增加一个足够的数量的板块将被化验到一个浅水库上的自动化液体处理。</li>
		<li>通过将氯化钠溶液的4.5 µL 转移到黑色384井存储板中的样品, 调整样品的盐浓度, 使其与标准曲线相匹配。</li>
		<li>将样品混合三次, 使用30µL 的混合容积, 并根据检测板的布局将样品的2µL 转移到白色浅层试片中。<ol>
<li>在样品板之间更改提示。</li>
		</ol>
</li>
		<li>室温下在黑暗中孵化1小时。</li>
		<li>对于要运行的检测板数量, 在检测缓冲区中制作足够数量的受体珠子。使用200µL 的二次受体珠的比例到12.3 毫升的化验缓冲器。</li>
		<li>使用多通道 pipettor, 将二次珠添加到黑色的384井存储板上。<ol>
<li>根据检测板布局, 将二次珠添加到指定用于样品和标准曲线的井中。</li>
			<li>为要运行的检测板数量添加足够数量的每井容积。</li>
		</ol>
</li>
		<li>使用自动液体处理程序, 将二次珠的10µL 转移到白色384井浅层测井板上。<ol>
<li>只添加到接受样品和标准曲线的水井, 根据化验板布局, 并清洗每个板块之间的提示。</li>
		</ol>
</li>
		<li>在黑暗中的室温下孵化一个小时。</li>
	</ol>
</li>
	<li>用透明的印版密封和扫描用与该检测相兼容的平板读卡器来密封检测板。使用0.2 毫米的板和探测器之间的距离, 180 毫秒的励磁时间, 和550毫秒测量时间的扫描。</li>
	<li>将原始数据从 IL-8 检测导入电子表格。</li>
	<li>使用 IL-8 检测标准曲线的自动曲线拟合, 然后将原始数据转换为每个样本的 pg/mL。</li>
</ol>

<ol>
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作者没有什么可透露的。
免责声明: 本文所表达的观点是作者的意见, 不反映陆军部、国防部或美国政府的官方政策。这项研究得到了 NIH/NIAID 与 USAMRICD 之间的一项跨部门协议的支持, 部分是由橡树管理的美国陆军医学研究所的研究生研究参与计划所提供的。通过美国能源部和 USAMRMC 之间的跨部门协议, 为科学和教育脊研究所。

在此, 我们描述了我们的方法和结果, 开发的高通量角膜上皮细胞筛选模型的研究 HF 和 CP 损伤。我们还从主 siRNA 屏幕上显示 HF 损伤的结果。研究 TIC 损伤的高温超导模型的发展面临着许多挑战。我们在文献中发现的与 HF、高放或 CP 在细胞培养模型中的研究有关的方法是没有帮助的。大多数的离体研究氟离子涉及口服细胞和使用氟化钠, 而不是 HF 或高放 (一些例子, 见28...

毒物性眼外伤是一种真正的眼部急症, 因为化学物质有可能迅速造成严重的组织损伤。不幸的是, 对毒物引起的角膜损伤的治疗只是普遍支持, 因为没有具体的治疗方法来治疗这些损伤。目前的治疗策略是非特定的, 主要包括局部治疗治疗, 如润滑剂, 抗生素, cycloplegics 后, 炎 (如类固醇), 一旦角膜重新表皮1 ,2。尽管现有最佳治疗方案可供选择, 但长期预后通常较差, 因为渐进性角膜混浊和新生血管2,3。
动物模型历来被用来研究化学毒性和理解损伤机制。然而, 动物研究是费时和昂贵的。还有努力减少动物试验。例如, 在欧洲联盟中达成立法 (欧共体 1907/2006) 有旨在减少动物试验的规定。这些规定包括要求公司共享数据, 以便在对动物进行建议的试验之前避免动物试验并获得欧洲化学品机构的批准。根据达成的规定, 动物试验应该是最后的手段。还有欧洲化妆品条例 (EC 1223/2009), 逐步淘汰动物化妆品的测试。当进行动物研究时, 它们遵循3Rs 的原则 (提炼、减少和替换), 这为执行更人性化的动物研究、减少使用的动物数量和使用非动物替代品提供了框架。尽可能。出于这些原因, 毒理学领域试图采用体外检测, 可以提供对毒性分子机制的洞察力, 并可以在更高的吞吐量4中进行。这是一种功能毒物学方法, 毒物是由它们的功能定义的, 而不是完全由它们的化学组成。更进一步, 功能致毒学寻求了解特定基因在毒物5的作用中所起的作用。随着 siRNA 技术的应用, 筛查在分子和细胞对毒物反应中的基因功能可以在高通量的情况下进行。siRNA 是双链 RNA 分子, 是19-25 核苷酸长, 利用后转录基因沉默路径存在的所有哺乳动物细胞6。这些都是综合制作和设计的目标一个特定的基因。当引入细胞时, siRNA 被处理, 一个链, 导链, 被加载到 RNA 诱导的沉默复杂 (RISC)。siRNA 将 risc 定向到 mrna 分子中的互补区域, risc 会降低 mrna 的降解。这导致了特定基因表达的减少。因此, 在毒物暴露的细胞中, 可以对特定基因表达的减少进行研究, 以确定该基因在细胞对毒物的反应中的作用。这种方法已被用来进一步了解蓖麻毒素的敏感性机制和 AHR 依赖性诱导 CYP1A17,8。
《化学恐怖主义风险评估 (CTRA) 清单》和《有毒工业化学品 (TIC) 》列出了根据其毒性和可能在恐怖主义、战争或工业事故事件9中释放的可能性对化学品进行分项的分类。我们正在应用一种 siRNA 高通量筛选 (高温超导) toxicogenomic 方法研究 CTRA 清单毒物, 这些有毒物质被认定在恐怖事件中有很高的使用风险。传统毒理学试图了解化学品对生物体的有害影响;然而, 我们还希望了解伤害机制, 以便向治疗和治疗方法的发展提供通知, 并有可能发现可作为疗法发展目标的分子。这种努力在某种程度上可以被认为类似于在药物发现过程中使用高通量 siRNA 筛选和细胞检测方法10。一个主要的区别是, 药物发现通常寻求一个单一的治疗发现的目标, 而在我们的方法, 它是有点不太可能有一个单一的目标, 具有较高的治疗价值的治疗毒物暴露。我们预计, 任何有效的毒物暴露治疗范式都需要一种多层面的方法来达到高治疗价值, toxicogenomic 数据可能会非常有效地告知一种行之有效的治疗范式。
台式自动化为制药或生物技术行业以外的实验室带来了高吞吐量方法。在我院的体外研究历来是传统的化验, 是低吞吐量11,12,13。在过去的几年里, 我们的实验室已经过渡到使用台式机器人进行高通量 siRNA 筛选。在此, 我们提出了眼部细胞模型的细化和对氟化氢 (HF) 和氯化苦 (CP) 的体外暴露方法的发展, 适合高通量 siRNA 筛查。我们的目标是确定分子, 以应对这些有毒物质来调节细胞损伤。我们选择的 siRNA 库的目标包括 G 蛋白耦合受体、蛋白激酶、蛋白酶、磷酸、离子通道和其他潜在的 druggable 靶。HF 和 CP 被选择为研究通过交叉引用 CTRA 名单代理与 ToxNet 报告的工业事故, 以找到那些目前最危险的眼睛伤害通过蒸气暴露9,14。CP (化学配方 Cl3CNO2, CAS 号 76-06-2) 最初被用作催泪瓦斯在一战15。它目前被用作农业熏蒸和杀线虫剂、杀菌剂和杀虫剂16的功能。氟化氢 (HF) 用于炼油厂的烷基化反应和有机化合物的电化学氟化物17。hf (化学方程式 hf, CAS 号 62778-11-4) 是一种气体, 但它的水形式是氢氟酸 (高放, cas 号 7664-39-3)。因此, 我们选择使用高放在我们的细胞暴露模型。选择 SV40 大 T 抗原永生化人角膜上皮细胞系 SV40-HCEC 进行研究。细胞活力和炎症标记 IL-8 被选择为终点, 因为参与细胞损伤的靶点应反映在细胞死亡和炎症反应中。具体地说, 如果目标在毒物暴露中起到保护作用, 当目标表达被 siRNA 抑制时, 细胞死亡和/或炎症细胞因子的产生就会增加。对于那些发挥消极作用的目标, 恰恰相反。此外, 慢性炎症在角膜暴露后的病理中似乎起到了作用, 而细胞死亡通路的干预可能会改善临床结局2,18。

<table><tbody><tr><td>Bravo liquid handing platform</td><td>Agilent or equivalent</td><td>G5409A</td><td></td></tr><tr><td>Bravo plate shaker</td><td>Agilent or equivalent</td><td>Option 159</td><td></td></tr><tr><td>Bravo 96LT disposable tip head</td><td>Agilent or equivalent</td><td>Option 178</td><td>96-channel large tip pipetting head unit</td></tr><tr><td>Bravo 96ST disposable tip head</td><td>Agilent or equivalent</td><td>Option 177</td><td>96-channel small tip pipetting head unit</td></tr><tr><td>Bravo 384ST disposable tip head</td><td>Agilent or equivalent</td><td>Option 179</td><td>384-channel small tip pipetting head unit</td></tr><tr><td>Bravo 96 250 &amp;mu;L sterile barrier tips</td><td>Agilent or equivalent</td><td>19477-022</td><td></td></tr><tr><td>Bravo 384 30 &amp;mu;L sterile barrier tips</td><td>Agilent or equivalent</td><td>19133-212</td><td></td></tr><tr><td>Bravo 384 70 &amp;mu;L sterile barrier tips</td><td>Agilent or equivalent</td><td>19133-212</td><td></td></tr><tr><td>EnSpire multimode plate reader</td><td>Perkin Elmer or equivalent</td><td>2300-0000</td><td>AlphaLISA assay detector with high power laser excitation</td></tr><tr><td>IL-8 (human) AlphaLISA Detection Kit&amp;nbsp;</td><td>Perkin Elmer or equivalent</td><td>AL224F</td><td>no-wash bead-based assay</td></tr><tr><td>ProxiPlate-384 Plus white 384-shallow well microplates</td><td>Perkin Elmer or equivalent</td><td>6008359</td><td></td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Invitrogen or equivalent</td><td>13778500</td><td>Transfection reagent</td></tr><tr><td>Opti-MEM 1 Reduced Serum Medium</td><td>Invitrogen or equivalent</td><td>31985070</td><td></td></tr><tr><td>TrypLE Express</td><td>Gibco or equivalent</td><td>12605010</td><td>Cell detachment solution</td></tr><tr><td>IncuCyte Zoom</td><td>Essen Instruments or equivalent</td><td>ESSEN BIOSCI 4473</td><td>Incubator-housed automated microscope</td></tr><tr><td>Chloropicrin</td><td>Trinity Manufacturing or equivalent</td><td>N/A</td><td>Acute toxicity and irritant</td></tr><tr><td>DMEM-F12 cell culture medium</td><td>Invitrogen or equivalent</td><td>11330-057</td><td>Contains HEPES</td></tr><tr><td>Fetal bovine serum</td><td>Invitrogen or equivalent</td><td>1891471</td><td></td></tr><tr><td>Human epidermal growth factor (cell culture grade)</td><td>Invitrogen or equivalent</td><td>E9644-.2MG</td><td></td></tr><tr><td>Recombinant human insulin (cell culture grade)</td><td>Invitrogen or equivalent</td><td>12585-014</td><td></td></tr><tr><td>Penicillin-Streptomycin solution (cell culture grade)</td><td>Invitrogen or equivalent</td><td>15140122</td><td></td></tr><tr><td>Hydrocortisone (cell culture grade)</td><td>Sigma or equivalent</td><td>H0888-10G</td><td></td></tr><tr><td>Glucose&amp;nbsp; (cell culture grade)</td><td>Sigma or equivalent</td><td>G7021</td><td></td></tr><tr><td>PBS&amp;nbsp; (cell culture grade)</td><td>Sigma or equivalent</td><td>P5493</td><td></td></tr><tr><td>siRNA</td><td>Dharmacon or equivalent</td><td>various</td><td></td></tr><tr><td>Thiazolyl blue tetrazolium bromide</td><td>Sigma or equivalent</td><td>M5655</td><td>MTT assay substrate</td></tr><tr><td>siRNA buffer</td><td>Thermo or equivalent</td><td>B002000</td><td></td></tr><tr><td>96-well cell culture plates</td><td>Corning or equivalent</td><td>CLS3595</td><td></td></tr><tr><td>T150 cell culture flasks</td><td>Corning or equivalent</td><td>CLS430825</td><td></td></tr><tr><td>BSL-2 cell culture hood</td><td>Nuaire or equivalent</td><td>NU-540</td><td></td></tr><tr><td>300 mL robotic reservoirs</td><td>Thermo or equivalent</td><td>12-565-572&amp;nbsp;</td><td></td></tr><tr><td>96 baffled automation reservoirs</td><td>Thermo or equivalent</td><td>1064-15-8</td><td></td></tr><tr><td>500 mL sterile disposable storage bottles</td><td>Corning or equivalent</td><td>CLS430282</td><td></td></tr><tr><td>Microplate heat sealer</td><td>Thermo or equivalent</td><td>AB-1443A</td><td></td></tr><tr><td>Microplate heat sealing foil</td><td>Thermo or equivalent</td><td>AB-0475</td><td></td></tr><tr><td>Cardamonin</td><td>Tocris or equivalent</td><td>2509</td><td>Anti-inflammatory, used as positive control</td></tr><tr><td>SKF 86002&amp;nbsp;</td><td>Tocris or equivalent</td><td>2008</td><td>Anti-inflammatory, used as positive control</td></tr><tr><td>DMSO</td><td>Sigma or equivalent</td><td>D8418</td><td></td></tr><tr><td>48% hydrofluoric acid</td><td>Sigma or equivalent</td><td>339261</td><td>Corrosive and acute toxicity</td></tr><tr><td>1000 &amp;mu;L Single channel pipettors</td><td>Rainin or equivalent</td><td>17014382</td><td></td></tr><tr><td>200 &amp;mu;L Single channel pipettors</td><td>Rainin or equivalent</td><td>17014391</td><td></td></tr><tr><td>20 &amp;mu;L Single channel pipettors</td><td>Rainin or equivalent</td><td>17014392</td><td></td></tr><tr><td>1000 &amp;mu;L 12-channel pipettors</td><td>Rainin or equivalent</td><td>17014497</td><td></td></tr><tr><td>200 &amp;mu;L 12-channel pipettors</td><td>Rainin or equivalent</td><td>17013810</td><td></td></tr><tr><td>20 &amp;mu;L 12-channel pipettors</td><td>Rainin or equivalent</td><td>17013808</td><td></td></tr><tr><td>Pipettor tips 1000 &amp;mu;L</td><td>Rainin or equivalent</td><td>17002920</td><td></td></tr><tr><td>Pipettor tips 200 &amp;mu;L</td><td>Rainin or equivalent</td><td>17014294</td><td></td></tr><tr><td>Pipettor tips 20 &amp;mu;L</td><td>Rainin or equivalent</td><td>17002928</td><td></td></tr><tr><td>Chemical fume hood</td><td>Jamestown Metal Products</td><td>MHCO_229</td><td></td></tr><tr><td>384-well sample storage plates</td><td>Thermo or equivalent</td><td>262261</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma or equivalent</td><td>S6191</td><td></td></tr><tr><td>50 mL conical tubes</td><td>Thermo or equivalent</td><td>14-959-49A</td><td></td></tr><tr><td>Serological pipettes 50 mL</td><td>Corning or equivalent</td><td>07-200-576</td><td></td></tr><tr><td>Serological pipettes 25 mL</td><td>Corning or equivalent</td><td>07-200-575</td><td></td></tr><tr><td>Serological pipettes 10 mL</td><td>Corning or equivalent</td><td>07-200-574</td><td></td></tr><tr><td>Serological pipettes 5 mL</td><td>Corning or equivalent</td><td>07-200-573</td><td></td></tr><tr><td>SV40-HCEC immortalized human corneal epithelial cells</td><td>N/A</td><td>N/A</td><td>These cells are not commercially available, but can be obtained from the investigators cited in the article</td></tr><tr><td>Sceptor Handheld Automated Cell Counter</td><td>Millipore or equivalent</td><td>PHCC20060</td><td></td></tr><tr><td>GeneTitan Multi-Channel (MC) Instrument</td><td>Affymetrix or equivalent</td><td>00-0372</td><td></td></tr><tr><td>Affymetrix 24- and 96-array plates</td><td>Affymetrix or equivalent</td><td>901257; 901434</td><td></td></tr><tr><td>Draegger tube HF</td><td>Draeger or equivalent</td><td>8103251</td><td></td></tr><tr><td>Draegger tube CP</td><td>Draeger or equivalent</td><td>8103421</td><td></td></tr><tr><td>Draegger pump</td><td>Draeger or equivalent</td><td>6400000</td><td></td></tr><tr><td>Clear Plate seals</td><td>Resesarch Products International or Equivalent</td><td>202502</td><td></td></tr><tr><td>Reagent reservoirs</td><td>VistaLab Technologies or equivalent</td><td>3054-1000</td><td></td></tr><tr><td>Xlfit</td><td>IDBS or equivalent</td><td>N/A</td><td>Excel add-in used for automated curve fitting</td></tr></tbody></table>

high throughput sirna screening for chloropicrin and hydrogen fluoride-induced cornea epithelial cell injury

毒物性眼外伤是一种真正的眼部急症, 因为化学物质有可能迅速造成严重的组织损伤。毒物性角膜损伤的治疗通常是支持的, 因为没有具体的治疗方法来治疗这些损伤。在努力开发治疗和治疗, 以照顾暴露, 这是很重要的了解分子和细胞机制的这些伤害。我们建议利用高通量小抑制 RNA (siRNA) 筛查可以是一个重要的工具, 有助于更迅速地阐明分子机制的化学角膜上皮损伤。siRNA 是双链 RNA 分子, 是19-25 核苷酸长, 利用后转录基因沉默途径降解 mRNA, 有同源性的 siRNA。因此, 在毒物暴露的细胞中, 可以对特定基因表达的减少进行研究, 以确定该基因在细胞对毒物的反应中的作用。本文介绍了氟化氢 (HF) 和氯化苦 (CP) 致眼损伤的高吞吐量筛查模型和方法的建立和验证。尽管我们选择了这两种毒物, 但我们的方法也适用于对有毒暴露协议进行细微修改的其他毒物的研究。选择 SV40 大 T 抗原永生化人角膜上皮细胞系 SV40-HCEC 进行研究。在筛选协议中选择了细胞活力和 IL-8 生产作为终点。介绍了与高温超导研究相适应的毒物暴露和细胞培养方法的一些挑战。这些毒物的高温超导模型的建立, 使进一步的研究, 以更好地了解损伤机制和筛查潜在治疗化学性眼外伤。

曝光方法开发
我们对人角膜上皮细胞系 SV40-HCEC 在高温超导研究中的适用性进行了改进和评价。SV40-HCEC 是永生化使用 SV-40 大 T 抗原, 是一个礼物从 Dhanajay Pal23。在曝光方法论的发展中, 有太多的变数被探索出来, 因此, 只有一些我们认为可以更广泛地适用于多种毒物的结果样本。
<p class="jove_cont...

Watch this Scientific Journal Video about High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury at JoVE.com

High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

高通量小抑制 RNA 筛选是一种重要的工具, 有助于更迅速地阐明化学角膜上皮损伤的分子机制。本文对氟化氢-氯化苦诱导的角膜上皮损伤进行高通量筛选的暴露模型和方法进行了开发和验证。

高通量 SiRNA 筛选氯化苦和氟化氢引起的角膜上皮细胞损伤

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments ...

high-throughput-sirna-screening-for-chloropicrin-hydrogen-fluoride

Research

JoVE Journal

Biology

5.8K 次观看.  US Army Medical Research Institute of Chemical Defense. 高通量小抑制 RNA 筛选是一种重要的工具, 有助于更迅速地阐明化学角膜上皮损伤的分子机制。本文对氟化氢-氯化苦诱导的角膜上皮损伤进行高通量筛选的暴露模型和方法进行了开发和验证。

视频: High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group. Scratch-induced directional wounding assays, and western blotting, and flow cytometry were then performed. We conclude that HSP27 has roles in corneal epithelial wound healing that may involve epithelial cell apoptosis and migration. Here, step-by-step descriptions of sample preparation and the study protocol are provided.

Herein, we present a protocol to use heat shock protein 27 (HSP27)-specific small interfering RNA to assess the function of HSP27 during corneal epithelial wound healing. RNA interference is the best method for effectively knocking-down gene expression to investigate protein function in various cell types.

角膜上皮伤口愈合过程中的热休克蛋白27的功能为基础的RNA干扰研究

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a ...

科研

遗传学

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

腺苷酸环化酶的药物引起的过敏性：含量简化和小型化的小分子和siRNA筛选应用

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

生物工程

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

高通量筛选RNA病毒的广谱化学抑制剂

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

免疫与感染

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

发育生物学

Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and throughput of cell culture. Mature human retinal organoids are utilized to screen potential pharmaceutical treatments for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).
We have recently shown that MacTel can be caused by elevated levels of an atypical lipid species, deoxysphingolipids (deoxySLs). These lipids are toxic to the retina and may drive the photoreceptor loss that occurs in MacTel patients. To screen drugs for their ability to prevent deoxySL photoreceptor toxicity, we generated human retinal organoids from a non-MacTel induced pluripotent stem cell (iPSC) line and matured them to a post-mitotic age where they develop all of the neuronal lineage-derived cells of the retina, including functionally mature photoreceptors. The retinal organoids were treated with a deoxySL metabolite and apoptosis was measured within the photoreceptor layer using immunohistochemistry. Using this toxicity model, pharmacological compounds that prevent deoxySL-induced photoreceptor death were screened. Using a targeted candidate approach, we determined that fenofibrate, a drug commonly prescribed for the treatment of high cholesterol and triglycerides, can also prevent deoxySL toxicity in the cells of the retina.
The toxicity screen successfully identified an FDA-approved drug that can prevent photoreceptor death. This is a directly actionable finding owing to the highly disease-relevant model tested. This platform can be easily modified to test any number of metabolic stressors and potential pharmacological interventions for future treatment discovery in retinal diseases.

Here we present a step-by-step protocol to generate mature human retinal organoids and utilize them in a photoreceptor toxicity assay to identify pharmaceutical candidates for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).

用于药物发现的人视网膜类器官毒性筛选

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and ...

在肝癌细胞中使用二氢乙锭进行新型 ROS 定量

High Throughput Screening Assessment of Reactive Oxygen Species (ROS) Generation using Dihydroethidium (DHE) Fluorescence Dye

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS production plays a central role in the spatial and temporal modulation of normal cellular functions such as proliferation, signaling, apoptosis, and senescence. In contrast, chronic ROS overproduction is responsible for a wide spectrum of diseases, such as cancer, cardiovascular disease, and diabetes, among others. Quantifying ROS levels in an accurate and reproducible manner is thus essential to understanding normal cellular functionality. Fluorescence imaging-based methods to characterize intra-cellular ROS species is a common approach. Many of the imaging ROS protocols in the literature use 2&#39;-7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) dye. However, this dye suffers from significant limitations in its usage and interpretability. The current protocol demonstrates the use of a dihydroethidium (DHE) fluorescent probe as an alternative method to quantify total ROS production in a high-throughput setting. The high throughput imaging platform, CX7 Cellomics, was used to measure and quantify the ROS production. This study was conducted in three hepatocellular cancer cell lines - HepG2, JHH4, and HUH-7. This protocol provides an in-depth description of the various procedures involved in the assessment of ROS within the cells, including - preparation of DHE solution, incubation of cells with DHE solution, and measurement of DHE intensity necessary to characterize the ROS production. This protocol demonstrates that DHE fluorescent dye is a robust and reproducible choice to characterize intracellular ROS production in a high-throughput manner. High throughput approaches to measure ROS production are likely to be helpful in a variety of studies, such as toxicology, drug screening, and cancer biology.

作者聚焦：肝细胞系中细胞内 ROS 水平的高通量测量

This protocol describes a novel method to quantify intracellular reactive oxygen species (ROS) using dihydroethidium (DHE) as a fluorescence dye probe using a high-throughput screening approach. The protocol describes the methods for quantitative assessment of intracellular reactive oxygen species (ROS) in the three different hepatocellular carcinoma cell lines.

使用二氢乙锓 （DHE） 荧光染料生成活性氧 （ROS） 的高通量筛选评估

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

生物学

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

高通量 SiRNA 筛选氯化苦和氟化氢引起的角膜上皮细胞损伤

摘要

探索更多视频

此视频中的章节

角膜上皮伤口愈合过程中的热休克蛋白27的功能为基础的RNA干扰研究

腺苷酸环化酶的药物引起的过敏性：含量简化和小型化的小分子和siRNA筛选应用

高通量筛选RNA病毒的广谱化学抑制剂

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C）损伤的反应

用于药物发现的人视网膜类器官毒性筛选

使用二氢乙锓（DHE）荧光染料生成活性氧（ROS）的高通量筛选评估

使用二氢乙锓（DHE）荧光染料生成活性氧（ROS）的高通量筛选评估

使用二氢乙锓（DHE）荧光染料生成活性氧（ROS）的高通量筛选评估

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

高通量筛选转录后调控基因的化学调节剂

高通量 SiRNA 筛选氯化苦和氟化氢引起的角膜上皮细胞损伤

摘要

探索更多视频

此视频中的章节

角膜上皮伤口愈合过程中的热休克蛋白27的功能为基础的RNA干扰研究

腺苷酸环化酶的药物引起的过敏性：含量简化和小型化的小分子和siRNA筛选应用

高通量筛选RNA病毒的广谱化学抑制剂

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

用于药物发现的人视网膜类器官毒性筛选

使用二氢乙锓 （DHE） 荧光染料生成活性氧 （ROS） 的高通量筛选评估

使用二氢乙锓 （DHE） 荧光染料生成活性氧 （ROS） 的高通量筛选评估

使用二氢乙锓 （DHE） 荧光染料生成活性氧 （ROS） 的高通量筛选评估

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

高通量筛选转录后调控基因的化学调节剂

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C）损伤的反应

使用二氢乙锓（DHE）荧光染料生成活性氧（ROS）的高通量筛选评估

使用二氢乙锓（DHE）荧光染料生成活性氧（ROS）的高通量筛选评估

使用二氢乙锓（DHE）荧光染料生成活性氧（ROS）的高通量筛选评估